Primary open-angle glaucoma (POAG) is a progressive disease leading to optic nerve damage and, ultimately, loss of vision. The cause of this disease has been the subject of extensive studies for many years, but is still not fully understood. Glaucoma results in the neuronal degeneration of the retina and optic nerve head, a gradual loss of retinal ganglion cells (“RGCs”), a decline of visual function, and ultimately blindness [Clark et al., Nature Reviews Drug Discovery, 2003, Vol. 2(6):448-459].
Several theories have been proposed to elucidate the etiology of glaucoma. One theory suggests that excessive intraocular pressure (IOP), which in some cases may be coupled with genetic defects on the optic nerve head, disrupts the normal axonal transport along the optic nerve and leads to RGC injury. Glaucoma treatment via reduction in IOP is frequently achieved with prescription eye drops containing therapeutic agents that suppress aqueous humor production. These agents include beta-blockers, such as timolol and betaxolol, and carbonic anhydrase inhibitors, such as dorzolamide and brinzolamide. Recently the use of prostaglandin analogs, such as latanoprost, bimatoprost and travoprost, which are believed to reduce IOP by increasing uveoscleral outflow, has become common. In cases where drug therapy is ineffective, treatment with lasers or surgery to reduce IOP may be required [Lee et al., Am. J. Health Syst Pharm., 2005, Vol. 62(7):691-699].
Disturbance of axonal transport of the optic nerve hinders traffic of intracellular molecules between the RGC soma and its terminal. Among the intracellular molecules of importance are neurotrophic factors. Neurotrophic factors are peptide molecules which stimulate or otherwise maintain growth of neural tissue. The transport of neurotrophic factors from the brain to the cell body of RGCs is essential to the survival of the RGCs. Deprivation of neurotrophic factors can induce apoptosis of neurons, and may be a cause of glaucoma-induced RGC apoptosis; see for example: Kuehn et al., Ophthalmol. Clin. North Am., 2005, Vol. 18(3):383-395; Anderson et al., Invest. Ophthalmol., 1974, Vol. 13(10):771-783; Quigley et al., Invest. Ophthalmol., 1976, Vol. 15(8):606-616; Mansour-Robaey et al., Proc. Natl. Acad. Sci. USA, 1994, Vol. 91(5):1632-1636; Meyer-Franke et al., Neuron 1995, Vol. 15(4):805-819.
The neurotrophin (“NT”) family of peptides include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4/5 and NT-6. They act by binding to neuron surface receptors, such as TrkA, TrkB, and TrkC. The Trk receptors are tyrosine kinases. TrkA is selective for NGF, TrkB is selective for both BDNF and NT-4/5, whereas TrkC is selective for NT-3. After binding, the NT-receptor complex is internalized and transported via the axon to the soma. These receptors undergo ligand-induced phosphorylation and dimerization, and activate a cascade of Ras protein-mediated signal transduction events that affect multiple vital functions of the neuron (Lewin et al., Ann. Rev. Neurosci., 1996, Vol. 19:289-317; Segal, R. A., Annu. Rev. Neurosci., 2003, Vol. 26:299-330; Kaplan et al., Curr. Opin. Cell Biol., 1997, Vol. 9(2):213-221]. Thus, these receptors play a fundamental role in the regulation of survival and differentiation of developing neurons and contribute to the maintenance of neuronal machinery in adult life.
The EgLN enzyme family are 2-oxoglutarate-dependent prolyl hydroxylases that catalyze the constitutive hydroxylation of the HIF-1α protein under normoxic conditions [Peso et al., J. Biol. Chem., 2003, Vol. 278(49):48690-48695; Ivan et al., PNAS, 2002, Vol. 99(21):13459-13464]. The hydroxylated HIF-1α protein is targeted for polyubiquination and proteasomal degradation by pVHL, the protein product of the von Hippel-Landau gene. Under hypoxic conditions, oxygen concentration becomes rate-limiting and EgLN-catalyzed hydroxylation is inefficient. Consequently HIF-1α escapes destruction and forms a heterodimer with HIF-1β. The complex is transported to the nucleus, where it acts as a transcription factor to up-regulate production of hypoxia-induced proteins and growth factors, such as vascular endothelial growth factor (VEGF).
The EgLN-3 isozyme also appears to be involved as an effector of apoptosis in sympathetic neurons under certain conditions. In particular, EgLN-3 is a downstream effector of nerve growth factor (NGF) withdrawal-induced apoptosis in NGF-dependent neurons. Expression of SM-20, a rat ortholog of EgLN-3, increases after NGF withdrawal in sympathetic neurons [Lipscomb et al., J. Neurochem. 1999, Vol. 73(1)429-432]. Induced expression of SM-20 causes apoptosis in sympathetic neurons even in the presence of NGF in a caspase-dependent process [J. Neurochem., 2003, Vol. 85(2):318-328]. Although SM-20 is normally resident in the mitochrondria, a truncated form that localizes to the cytoplasm due to loss of a mitochondrial targeting sequence still induces apoptosis [Lipscomb et al., J Biol. Chem. 2001, Vol. 276(7):5085-5092].
These findings have recently been extended to developing neurons [Lee et al., Cancer Cell, 2005, Vol. 8:155-167]. During embryogenesis, sympathetic neuronal precursor cells that fail to make synaptic connections are starved of NGF and undergo c-Jun-dependent apoptosis [Schlingensiepen et al., Cell. Mol. Neurobiol., 1994, Vol. 14:487-505]. The risk of a type of neuronal cancer called familial pheochromocytoma is increased by germline mutations that inactivate pVHL or NF1 (an antagonist of the NGF receptor TrkA), or that activate c-RET (the receptor for glial derived neurotrophic factor, which cross-talks with TrkA). In each of these cases the intracellular concentration of the c-Jun antagonist JunB increases, inhibiting apoptosis. Germline mutations that reduce the activity of succinate dehydrogenase (SDH) also increase familial pheochromocytoma risk. Succinate is a co-product of EgLN-3-catalyzed proline hydroxylation and feedback inhibits the enzyme, and thus needs to be removed by SDH for EgLN-3 prolyl hydroxylase activity. Sporadic pheochromocytoma due to somatic mutation in one of these genes is rare since apoptosis of “unconnected” sympathetic neuronal precursor cells is not important once embryogenesis is complete.
NGF withdrawal-induced apoptosis requires EgLN-3 proline hydroxylase activity. EgLN-3-induced cell death is not reduced by co-expression of JunB. Additionally, EgLN-3 expression knockdown by siRNA inhibits c-Jun induced cell death. These observations indicate that EgLN-3 is necessary and sufficient for NGF withdrawal-induced apoptosis, and acts downstream of c-Jun. The presumed protein target of EgLN-3-catalyzed proline hydroxylation that is important for apoptosis induction has not been identified, although it is suspected that pVHL's polyubiquination (and subsequent marking for proteasomal destruction) of a hyperphosphorylated form of atypical protein kinase C is responsible for pVHL's suppression of JunB.
Induction of neuronal apoptosis via c-Jun N-terminal kinase (JNK)-catalyzed phosphorylation of c-Jun has been implicated as a contributing factor to retinal ganglion cell (RGC) death in high IOP-induced glaucoma models in monkeys [Hashimoto et al., Brain Research, 2005, Vol. 1054(2):103-115] and rats [Quigley et al., Exp Eye Res. 2005, Vol. 80(5):663-670; Wang et al., J Neurosci Res., 2005, Vol. 82(5):674-678; Kwong et al., J. Exp. Eye. Res., 2006, Vol. 82(4):576-582]. It is not known how important NGF withdrawal is the pathological progression of RGC loss in POAG, nor whether EgLN-3 can act via the JNK pathway to induce neuronal cell death.